Editing Cleancap mRNA Invitro Transcription (section)

== <span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">High yield Production of mRNA</span> ==

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">10 mL transcription reaction mixture<br/>30 mM Tris (pH 8.1 at 37°C), 15 mM Mg2+ , 10 mM dithiothreitol (DTT), 2 mM spermidine, 0.01% (v/v) Triton X-100, 4 mM each NTP, 1 mL of PCR-generated DNA template, and 0.1 mg/mL of T7 RNAP</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">optimal concentration of Mg2+ is 15 ~45 mM<br/>After 2.5 h of incubation at 37°C, pyrophosphate, which forms during in vitro transcription reaction, was pelleted down by centrifugation, and additional Mg2+ should be added to the reaction.&nbsp;<br/>The reaction continued till 5 h.&nbsp;<br/>Size-exclusion FPLC based RNA purification<br/>RNA sample preparation for chromatographic purification<br/>Transcription mixtures for the S-box RNA were heated to 90°C for 5 min, immediately transferred to ice for 5 min and then pelleted down (centrifuged at 16, 000g for 10 min). The supernatant was collected and mixed as 1: 4 with Tris-EDTA buffer containing 8 M urea and 100 mM NaCl before loading onto size-exclusion FPLC (SEC) columns. Sample pre-load treatment was operated to remove the T7 RNAP, residual pyrophosphates and aggregates formed during in vitro transcription.</span>

<span style="color: rgb(37, 37, 37); font-family: sans-serif; font-size: 14px;">Denaturing chromatographic purification<br/>The glass columns (890/10 mm) and (xk16/1000 mm) were packed according to manufacturer’s specifications with gel filtration matrix (Superdex-75, Superdex-200) and equilibrated in a cold room (4°C) with several column volumes (CV) of specified buffer. The transcription mixture from the previous step was loaded directly onto the equilibrated SEC column, and the chromatography was performed at 1 mL/min flow rate, collecting 2 mL fractions. Fractions were analyzed by denaturing urea-PAGE. S-box RNA containing fractions were combined and concentrated to 0.5 mL using Millipore centrifugal filter units (EMD Millipore), washed twice with the appropriate buffer for chemical probing analysis. All experiments were performed at 4°C. The efficiency of proposed purification system was confirmed through SHAPE experiments for S-box RNA.</span>

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